UV/Vis (Ultraviolet and Visible) Spectroscopy Agilent 8453 Diode Array UV-Vis Spectrophotometer Varian Cary 5000 UV-Vis-NIR Spectrophotometer
To Do s Read Chapters 13 & 14. Complete the end-of-chapter problems, 13-1, 13-2, 13-3,13-6 and 13-8. Complete the end-of-chapter problems, 14-2, 14-5, 14-6, 14-7, 14-8, 14-9, 14-10, 14-12, 14-14, 14-16, 14-19 and 14-20.
Wavelength and Spectroscopy
What Molecular Changes Cause UV/Vis Absorptions? λ = 200 ~ 700 nm UV/Vis spectroscopy is used to detect the presence of certain functional groups called chromophores, especially in conjugate systemssuch as aromatics, dienes, polyenes, and α,β -unsaturated ketones Electronic excitation Absorption of UV or visible light occurs only when the energy of incident radiation is the same as that of an electronic transition in the molecule.
Electronic Transitions Only n π* and π π* transitions usually show up in 200-700 nm. Only limited set of organic functional groups show UV/Vis absorptions.
Instrument (double beam) Typical λ = 200 ~ 700 nm
The Beer s Law I A λ = log ( ref ) I sample A λ = ε λ x c x l I ref I sample > 1, thus A λ I ref I sample I ref I sample is aways positive. = 1 A λ = 0 = 10 A λ = 1 ε λ : molar absorptivity constant or extinction coefficient (l mol -1 cm -1 ) c : concentration (mol/l) l : path length (cm) I ref I sample I ref I sample = 100 A λ = 2 = 1000 A λ = 3
Instrument (Diode Array) Rugged, fast, and reliable
Solvents and Cuvettes for UV/Vis Spectroscopy
Typical UV/Vis Spectra O CH 3
Franck-Condon Principle Nuclear motion is negligible during the time required for an electronic transition. A B Excitation can occur from the ground state to any of the excited state vibrational levels. The lines overlap and a continuous broad band is observed.
Difference Spectroscopy UV absorption spectra and temperature -difference spectra of HO5dCyt in 0.02 M NaOH/NaHCO3 buffer at ph 10.5 at 25 C (A, dashed line), 75 C (A, solid line) and 75 25 C (B). Comparable spectra of dcyt in 0.02 M NaOH/NaHCO3 buffer at ph 7 at 25 C (C, dashed line), 75 C (C, solid line) and 75 25 C (D).
Isosbestic Points ε HA = ε A- HA A - Isosbestic points are usually a diagnostic for the presence of only two absorbing species.
Spectral Shift blue shift red shift
Polymeric Nucleic Acids Have an Increased Molar Absorptivity When They are Converted to Smaller Units
Common Organic Chromophores conjugated and non-conjugated
The Carbonyl Groups λmax 270 ~ 300 nm εmax 10 ~ 100, n π* O C and R R 1 R O C H!* c o n The two orbitals, n and π*, are orthogonal to each other and promotion of electron requires significant changes in geometry. The electronic transition must be coupled with vibration that allows some overlap between these orbitals.
Other Carbonyl Groups! max " max!* O C H 3 C CH 3 H 3 C O C Cl (hexane) (hexane) 274 235 15 53 R C O X n O C H 3 C NH 2 (water) 214 - O!* H 3 C C OEt (water) 204 60 C=O n (X) H 3 C O C OH (ethanolr) 204 41 n
Different Carbonyl Compounds and Their UV/Vis
Solvent Effects C OI H O!* R n
Aldehydes and Ketons
Unconjugated Alkenes
Conjugated Alkenes
Conjugation and Spectral Shifts
β-carotene
Diene Isomers λmax = 239 nm 7 6 5 4 PPM 3 2 1 0 λmax = 278 nm 7 6 5 4 PPM 3 2 1 0
Empirical Calculation of λmax for Conjugated Alkenes
Empirical Calculation - Examples
Empirical Calculation More Examples
Steric Hindrance and Spectral Shifts H 3 C CH 3 CH 3 CH 3 CH 3 CH 3 H 3 C CH 2 H 3 C CH 2! max " max 225 6.400 231 10,000 H 3 C CH 3 CH 3 CH 3 Twisted diene low conjugation λmax and εmax H 3 C CH 2
Conjugated Carbonyls
Empirical Calculation alkyl or ring residues
Empirical Calculation - Examples
Exocyclic Alkene Issue O 215 12 β-alkyl 3 x 18 δ+ alkyl 2 x 5 exocyclic C=C 2 x 30 extra conjugation 351 nm Observed 348 nm
Homoconjugation
Homoconjugation- More Examples
Solvent Effect In hexane λmax = 295 εmax = 50 In ethanol λmax = 255 εmax = 12,500 215 12 (β-alkyl) 30 (β-oh) 257nm
Charge Transfer(CT) Bands A + D A D h! A D CT complex
Intramolecular CT Bands O!-!+
Benzene Chromophore O H n π*
Solvent Effects
Benzene Bands Designation
Substituent Effects
Benzene Derivatives - Monosubstituted
ph Sensitive Derivatives O OH OH - O O OH OH - O
ph Titration of Phenol Isosbestic point
pka Measurement
Aromatic Amino Acids H 3 N R O O R = Phe OH Tyr H N Trp
Benzene Derivatives - Disubstituted The mono-substituted moiety with the largest red shift will dominate the absorption of a di-substituted benzene.
Para-substituted Benzenes If one substituent is e-withdrawing and the other is e-donating, a significant red-shift is observed due to the extended conjugation.
Disubstituted Anilines
Disubstituted Benzaldehydes
Disubstituted Benzoic Acids
Fused Ring Systems
Heterocyclic Aromatics
Steric Effect Cis & Trans
Steric Effect E & Z E E Z E Z Z! max 328 313 299 " max 56,200 30,600 29,500
Steric Effect - Biphenyls
Empirical Calculation Benzoyl Derivatives
Empirical Calculation Examples Br 246 (ketone) + 3 (o-ring) + 2 (m-br) HO CO 2 H 230 (acid) + 7 x 2 (m-oh) + 25 (p-oh) O! max = 251nm obs = 253 nm HO OH! max = 269nm obs = 270 nm
Tautomerization O OH 239 (14,100) N H N N OH 260 (2,200) N 257 (2,750)
Porphyrin Chromophore
Exciton Coupling
UV/Vis Analysis A single band (e = 10 ~ 100) in 250 ~ 300 nm, no major absorption in 200 ~ 250 nm. n π*, C=O (ketone and aldehyde), C=N. N=N, -NO2 etc Two bands (e = 1,000 ~ 10,000) > 200 nm. π π*, aromatics Bands (e = 10,000 ~ 20,000) > 210 nm. π π*, conjugated C=C and C=O Compounds that are highly colored A long-chain conjugated system A polycyclic aromatic system ph-depended absorption Ionizable group attached to chromophore